Biochemistry 1991, 30, 4143-4151
4143
Isotope Trapping and Positional Isotope Exchange with Rat and Chicken Liver Phosphoenolpyruvate Carboxykinasest Cheau-Yun Chen,* Yoshitake Sato,s and Vern L. Schramm* Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461 Received September I I, 1990; Revised Manuscript Received December 5, I990
ABSTRACT: Isotope-trapping studies of the enzymeMgGTP complex were carried out with rat liver cytosolic and chicken liver mitochondrial phosphoenolpyruvate carboxykinases. For the rat liver enzyme, MgGTP was partially trapped from both E-MgGTP and E-MgGTPSOAA complexes, consistent with a steady-state random mechanism. For the chicken liver enzyme, MgGTP was 100% trapped from the ESMgGTP-OAA complex, consistent with a steady-state ordered mechanism. The rate constants for the interaction of MgGTP with the free enzymes are approximately lo7 M-' s-I, somewhat lower than the diffusion limit for association. The dissociation rate for the enzymeMgGTP complexes is 26-92 s-l, reflecting a tightly bound complex with high commitment to catalysis in the presence of oxaloacetate. Positional isotope-exchange studies were also carried out with phosphoenolpyruvate carboxykinases from rat and chicken. No exchange of the Py-'80 in [/3y-180,y-1803]GTPto form [/3-'80,r-1803]GTPwas detected in the absence of oxaloacetate. In the presence of oxaloacetate, no positional isotope exchange of [/3y-'80,y-1803] G T P was detected during initial rate conditions. The results indicate that at least one of the products dissociates rapidly from the EMgGDP.PEP.C02 complex relative to the net rate of MgGTP formation from the E.MgGDP.PEP.C02 complex. A rapid equilibrium between the central complexes in which the P-phosphoryl of G D P is restricted with respect to torsional rotation cannot be excluded but is unlikely on the basis of the relative rates of catalysis and torsional rotation. The addition of Mn2+, an activator of phosphoenolpyruvate carboxykinase, did not influence the positional isotope-exchange results. The py-I8Oto p - l 8 0 exchange of [/3y-180,y-'803]GTP was not induced by oxalate as a substrate analogue of oxaloacetate. The results are consistent with a mechanism of direct, unidirectional phosphoryl transfer between G T P and oxaloacetate during initial reaction rate conditions.
Phosphoenolpyruvate carboxykinase (P-enolpyruvate carboxykinase) (EC 4.1.1.32) catalyzes the reversible reaction oxaloacetate
+ MgGTP -'
Mn*+
phosphoenolpyruvate
+ MgGDP + COz
The primary function of this enzyme in vertebrates is to catalyze the conversion of oxaloacetate to phosphoenolpyruvate in gluconeogenesis. The enzyme exists as cytosolic and mitochondrial isoenzymes. These distinct proteins from different cellular compartments do not cross-react immunochemically (Ballard & Hanson, 1969). The enzymes are encoded by different mRNAs and have only 63% amino acid identity (Hod et al., 1982; Weldon et al., 1990). However, they share similar kinetic properties, which include the decarboxylation of oxaloacetate to pyruvate and C 0 2 in the presence of GDP and activation by Mn2+ even in the presence of excess Mg2+ (Schramm et al., 1981). In rat liver, the enzyme is found exclusively in the cytosol (Cornell et al., 1986). In contrast, the enzyme is found only in the mitochondria in chicken liver (Watford et a]., 1981). In contrast to the knowledge of tissue specificity and expression of the enzymes (Beale et al., 1985; Weldon et al., +This work was supported by NIH Research Grant GM 36604. Some of this work was performed in the Department of Biochemistry, Temple University School of Medicine, Philadelphia, PA 19140. * Author to whom correspondence should be addressed. *Present address: Department of Biochemistry, Temple University School of Medicine, Philadelphia, PA 19140. 1 Present address: Department of Pharmacology, Research Laboratory, Nippon Shinyaku Co. Ltd.. Nishiohji Hachijo, Minamiku, Kyoto, 601 Japan.
0006-2960 I9 1 10430-4 143fi02.50I O
1990), the catalytic mechanisms are poorly understood. Kinetic and isotope-exchange experiments (Jomain-Baum & Schramm, 1978) indicated a steady-state random mechanism for the rat enzyme while NMR studies of the complexes of substrates with the chicken enzyme have indicated rapid association-dissociation between substrates and enzyme on the NMR time scale (Lee & Nowak, 1984). In the present studies, both the rat and chicken liver Penolpyruvate carboxykinases are characterized with respect to the rate constants for interaction with MgGTP, the metabolically significant metal-nucleotide triphosphate complex. Isotope-trapping experiments (Rose et al., 1974) are used to establish the relative rates of catalysis and MgGTP dissociation. We also quantitate the rate constants for phosphoryl transfer relative to the rate of product release and the rate of re-formation of MgGTP from the E.MgGDP.PEP-CO2 complex using positional isotope exchange (Midelfort & Rose, 1976; Rose, 1980a). MATERIALS AND METHODS Materials. [CY-~~PIGTP, [y-32P]GTP(triethylammonium salt), and [8-3H]GDP (trisodium salt) were purchased from New England Nuclear. [32P]P-enolpyruvatewas synthesized from oxaloacetate and [y-32P]GTPby using P-enolpyruvate carboxykinase. GTP and GDP were purchased from Pharmacia. HEPES, P-enolpyruvate, and oxaloacetate were purchased from Sigma. Triethanolamine was purchased from Fisher Scientific. Poly(ethy1enimine)-cellulose F precoated plastic sheets for thin-layer chromatography were purchased from E. Merck. Purification of Chicken Liver and Rat Liver P-enolpyruvate Carboxykinase. Rat liver P-enolpyruvate carboxykinase was 0 1991 American Chemical Societv
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Chen et al.
at saturating oxaloacetate and saturating M ~ [ L Y - ~ ~ P ] G T P purified from rat liver cytosol (Brinkworth et al., 1981) except that ITP-Sepharose was used in place of GTP-Sepharose. (P*max). Chicken liver P-enolpyruvate carboxykinase was extracted and During the 2-s incubation, a maximum of 81 pM each of purified from chicken liver mitochondria (Ash et al., 1990). GDP, HC03- + CO,, and P-enolpyruvate is formed. With a kinetic constant of 30-50 mM for HCOC at pH 8 (JoEnzyme Assay. The routine assay in the direction of Pmain-Baum & Schramm, 1978) the reverse exchange reaction enolpyruvate formation was carried out at 30 OC in a reaction is assumed to be negligible during this period. mixture containing 50 mM Hepes, pH 8.0, 10 mM MgCl,, 0.1 mM MnC12, 0.1 mM GTP, 4 mM L-malate, and 1 mM Synthesis of [@y-'80,y-1803] GTP. I80-Enriched inorganic NAD+. Reaction rates were based on the extinction coefficient phosphate was synthesized with PCl, and H2180according to of NADH of 6.22 mM-' cm-I at 340 nm. Malate dethe procedure of Hackney et al. (1980) with slight modificahydrogenase (1 l units) was added first to generate oxalotions. The synthesis was carried out in a sealed plastic bag acetate. The reaction was then initiated by adding P-enolunder N, evacuated to a NaOH trap. After 180-enrichedH 2 0 pyruvate carboxykinase. A unit of catalytic activity forms 1 (1 .O mL, 50 mmol) reacted with PCl, (1.79 g, 8.6 mmol), the pmol of P-enolpyruvate/min, with the routine assay described solution was neutralized with 4.2 mL of dry pyridine. The above. concentration of I80-enriched inorganic phosphate was determined according to Lanzetta et al. (1979). 31P NMR The assay in the direction of oxaloacetate formation was analysis indicated that the pyridine phosphate salt was 9 1% carried out at 30 OC by using a similar procedure except the I8O4enriched and 9% I8O3enriched. reaction mixture contained 50 mM Hepes, pH 8.0, 10 mM MgCl,, 0.1 mM MnCI,, 0.5 mM GDP, 0.5 mM P-enol[@y-180,y-1803]GTP was synthesized according to the pyruvate, 50 mM KHCO,, 0.14 mM NADH, and 11 units procedure of Webb (1980). 31PNMR analysis demonstrated of malate dehydrogenase. The reaction was initiated by the that the synthesized GTP was 90% I8O4 enriched in the yaddition of P-enolpyruvate carboxykinase. A unit of catalytic phosphorus and 10% 1 8 0 3 enriched. activity forms 1 pmol of oxaloacetate/min with this assay. Positional Isotope Exchange with [@y-180,y-'803]GTP. The protein concentration was determined by the method The positional isotope-exchange experiments were carried out of Bradford (1 976) with reagents purchased from Bio-Rad and with both rat and chicken P-enolpyruvate carboxykinase. For with bovine serum albumin as a standard. chicken liver P-enolpyruvate carboxykinase, the experiments were carried out with oxaloacetate either added directly or The K, for oxaloacetate was determined in a reaction produced from malate by the malate dehydrogenase reaction. mixture of 50 mM triethanolamine at pH 8.0, with 5 mM For rat liver P-enolpyruvate carboxykinase, the experiments MgCl,, 5 mM GTP, 50 pM MnC12, varied amounts of Lwere carried out with oxaloacetate added directly. malate, and 1 mM NAD+. Malate dehydrogenase (12 units) was added to establish the equilibrium with oxaloacetate. The When positional isotope-exchange experiments were carried difference of absorption at 340 nm was used to determine out with oxaloacetate produced from the malate dehydrogenase oxaloacetate concentration. The initial reaction rates were reaction, the oxaloacetate concentration was determined by determined as described above. NADH production. The activity of P-enolpyruvate carboxykinase was determined in the positional isotope-exchange Isotope Trapping of EsMgGTP. Isotope-trapping studies reaction mixture by measuring depletion of oxaloacetate were carried out at room temperature as described by Rose coupled to the malate dehydrogenase reaction. Typically, the (1980b). A 10-pL equilibrium solution contained 50 mM positional isotope-exchange reactions were carried out at 30 triethanolamine, pH 8.0, 0.5 mM MgCI,, 0.05 mM MnC12, OC in a mixture of 100 mM HEPES, pH 8.0, 8.5 mM MgCl,, and [ w ~ ~ P ] G Tand P P-enolpyruvate carboxykinase in the 3.5 mM [@y-180,y-'803]GTP, 1 mM NAD', 50 mM malate indicated amounts. For chicken liver P-enolpyruvate car(K+), and 11 units/mL malate dehydrogenase. After a 10-min boxykinase, 50 pM enzyme (based on MW = 72000; specific incubation at 30 "C, an aliquot (1 mL) was collected as a activity = 14.9 units/mg) was included in the equilibrium control. NAD+, malate, and malate dehydrogenase were not solution. For rat liver P-enolpyruvate carboxykinase, 55 pM included when the experiments were carried out in the absence enzyme (based on MW = 72000; specific activity = 14.9 of oxaloacetate. P-enolpyruvate carboxykinase was added last units/mg) was included. A 100-pL chase solution contained to initiate the reaction. At variable intervals, 1 mL of the 50 mM triethanolamine, pH 8.0,30 mM MgCl,, 30 mM GTP, reaction mixture was removed and added to 50 p L of CCl, and 50 pM MnCI2, and oxaloacetate concentration was varied and vortexed vigorously. After the precipitated protein was from 0.025 to 2 mM. The catalytic reaction was initiated by removed by centrifugation, the extent of the reaction was rapidly mixing the equilibrium mixture with the chase solution. determined by measuring the increase in NADH during the After 2 s, the reaction was terminated by the addition of formic reaction. The supernatant was then mixed with 25 mM acid to a concentration of 1.2 M. The precipitated protein EDTA, pH 9.0, and 40% D 2 0 (for field frequency locking), was removed by centrifugation. [CY-~~PIGDP was isolated from followed by 3'P NMR measurement. The GDP produced was [ c ~ - ~ ~ P ] GonT aP PEI-cellulose plate developed with 0.75 M potassium phosphate, pH 3.5. The amount of [ C Y - ~ ~ I G D P also quantitated by the ,'P NMR measurement based on the chemical shift of the @-phosphorusof GDP (0.33 ppm upfield formed was determined by scintillation counting. The controls relative to the y-phosphorus of GTP). were treated identically, except the same amount of [cY-~,]GTP used in the equilibrium solution was mixed in the chase solution When the experiments were carried out with oxaloacetate to correct for the amount of [cY-~~PIGDP produced from added directly, the oxaloacetate was dissolved immediately M~[cx-~*P]GTP following release from the E . M ~ [ c Y - ~ ~ P ] G T P before use at 0 OC and was spectrophotometrically calibrated complex and before stopping the reaction with formic acid. by the depletion of NADH in a reaction mixture containing The [CX-~~P]GDP formed in these control experiments was 100 mM HEPES (K'), pH 8.0, 0.22 mM NADH, and 6 negligible in comparison with the experimental value. The units/mL malate dehydrogenase. The activity of P-enolexperiments were repeated with varied amounts of [(u-~~PIGTP pyruvate carboxykinase in the reaction mixture for the posiin the equilibrium mixture to determine the dissociation tional isotope exchange was measured by the production of P-enolpyruvate coupled to the pyruvate kinase and lactate constant of E-MgGTP and the amount of [CX-~~PIGDP formed
kz
ki
!
lq
“The complex of E.M~[(U-~*P]GTP is formed in the equilibrium mixture before the addition of additional unlabeled MgGTP and oxaloacetate (OAA).
dehydrogenase reactions. The reaction mixture contained 100 mM HEPES (K+), pH 8.0, 18 mM MgC12, 5.0 mM GTP, 3.0 mM oxaloacetate (K+),1 mM ADP, 0.5 mM NADH, 100 mM KCI, 8 units/mL pyruvate kinase, and 15 units/mL lactate dehydrogenase. The results were corrected for the nonenzymatic decarboxylation of oxaloacetate. The activity of P-enolpyruvate carboxykinase was also determined in the presence of 50 pM MnC12 in an analogous procedure. Typical positional isotope-exchange reactions were carried out at 30 OC in reaction mixtures that contained 100 mM HEPES (K+), pH 8.0, 18 mM MgCI2, and 5.0 mM [by180,y-1803]GTP.Oxaloacetate at 3 mM was added immediately before the P-enol pyruvate carboxykinase to minimize nonenzymatic decarboxylation. At indicated intervals, the reaction was terminated by vortexing 1 mL of the samples with 50 p L of CCl,. Samples were then treated as described above. Positional isotope-exchange experiments with oxalate were the same, except that oxaloacetate was substituted by 1 mM oxalate. 3 1 PN M R Measurements. The 31Pspectra were measured on a Varian 500-MHz multinuclear spectrometer operating at 202.3 MHz by using a VXR-4000 data station. Spectra were recorded at 25 OC with a spectral width of 8000 Hz and an acquisition time of 2 s. Zero filling to 32000 was applied before Fourier transformation with a digital resolution of 0.489 Hz/data point. The samples were measured in a 10-mm probe, and 600-1000 scans were averaged to obtain an adequate signal-to-noise ratio. The chemical shifts were measured relative to K2HP04 as an external standard. The rate of positional isotope exchange was determined by the peak area corresponding to P[I8O4] and PO[’803]at the y-phosphoryl group of GTP. The fraction of the original [by-’80,y1803]GTPpool converted into GDP during the positional isotope-exchange experiment was determined by the peak area of the y-phosphorus of GTP (6 = -8.053 ppm, J = 19.1 Hz) relative to the P-phosphorus of GDP (6 = -8.386 ppm, J = 21.5 Hz). Exchange of [8-3H]GDPinto GTP. Reaction conditions were the same as the positional isotope-exchange experiment. After 40 min of reaction in the positional isotope-exchange reaction, a trace amount of [8-’H]GDP (1 pCi, lo-, pmol) was added to a 1-mL reaction mixture to follow the exchange reaction into GTP. At variable intervals, samples were terminated by adding 8 p L of the reaction mixture to 9 pL of 1 N formic acid. The GDP was separated from GTP on PEl-cellulose TLC, developed in 0.75 M KH2P04, pH 3.5. The [8-3H]GDPand [8-3H]GTPspots were excised, and the radioactivity was measured. Exchange of [32P]P-enolpyruuateinto GTP. The experiments were similar to that for [8-3H]GDP exchange, except that [€b3H]GDP was replaced by a trace amount of [32P]P-
enolpyruvate (0.3 pCi) in the exchange reaction mixture. The P-enolpyruvate was separated from GTP by using PEI-cellulose TLC, developed in 0.75 M KH2P04, pH 3.5. The Rr values for GTP and P-enolpyruvate are 0.22 and 0.79, respectively. The [32P]P-enolpyruvateexchange rate was based on the fraction of [32P]P-enolpyruvate exchanged and its concentration. Data Analysis. Results of isotope trapping were fitted with the rate equation (eq 1) and the programs developed by Cleland (1 979):
where P* is the amount of GTP* trapped at a given concentration of oxaloacetate varied in the chase. [OAA] represents the concentration of oxaloacetate. P*, represents the amount of [ w ~ ~ P ] G Ttrapped P when the oxaloacetate concentration is saturating. KbAA represents the concentration of oxaloacetate to give half of P*,,,. Results of P*maxat each fixed concentration of M ~ [ c Y - ~ ~ ] GinT the P enzyme-MgGTP equilibrium mixture were then analyzed by using -=-1(lt P*mx
1 P*max,sat
[AI - P*max
where [A] represents the concentration of M~[cY-’~P]GTP and P*,,, represents the amount of [ d 2 P ] G T P trapped at saturepresents the P*,,, when the rating oxaloacetate. P*max,sat enzyme is saturated with MgGTP in the equilibrium mixture, and Kd represents the dissociation constant for the E-MgGTP complex. As shown in Scheme I, the ratio of the rate of dissociation of M ~ [ c x - ~ ~ P ] Gfrom T P E.M~[cI-~~P]GTP-OAA (k6) relative (k’ll) can be estimated from to its conversion to [cx-~~PIGDP k6 _ - E . M ~ [ c ~ - ~ ~ P ] -G1T P k’ll
(3)
P*max
A limiting value for the dissociation rate of M ~ [ c ~ - ~ * P ] G T P from E . M ~ [ c Y - ~ ~ P ] G( kT2P)can be determined with (Rose, 1980b) K‘OAAVmax KOAA Et
KbAA [ E * M ~ [ w ~ ~ P ] G Vmax TP]
-- I k Z I -
KOAA
-
P*max
Et
(4)
where K o A A represents the concentration of oxaloacetate to give half Vmax/Et,Vmax/Etrepresents the turnover number of P-enolpyruvate carboxykinase in the direction of P-enolpyruvate formation, and K b A A and P*,,, are the same as described above.
4146
Biochemistry, Vol. 30, No. 17, 1991
Chen et al.
o.'2F---77
0.10
n
h n
CJ
Y \
l-
o'"[\
pM GTP trapped 50 pM GTP trapped
'
0.00 0
20
10
30
l/oxaloacetate
j . 0.05 \
V
l-
0.04
I
I 40
50
(l/mM)
I B
I
x
E"
O.O3
a &
0.02
2
0.01
n \
l-
0.00
0.00
0.05
0.10
1/([GTP*]-[GDP*]max)
'
0.1 5
0.00 0.00
(1/pM)
Isotope trapping from the E-M~[(u-~~P]GTP complex with rat liver P-enolpyruvate carboxykinase. (A) The [(U-~~PIGDP produced at varied oxaloacetate concentrations (0.025-2.0 mM) was determined as described in the text. Each line represents experiments beginning with a different concentration of M ~ [ ( u - ~ ~ P ] in GT theP equilibrium mixture. E s M ~ [ ~ - ~ ~ Pat ] Gdifferent T P concentrations was produced in the equilibrium solution by mixing 5 5 pM enzyme and Mg[w 32P]GTPat concentrations of ( 0 )24, (0)50, (m) 80, and (0)120 pM. The lines are the best fit of the data to eq 1. (B) The P*,,, ( [o-~*P]GDP produced at a saturating concentration of oxaloacetate) T Pequilibrium mixture determined with varied M ~ [ ( U - ~ ~ P ]inGthe was analzyed in the double-reciprocal plot of l/P*,,x vs I/(Mg[cr32P]GTP- P*,,,). The lines are the best fit of the data to eq 2. FIGURE 1:
The rate constants of the positional isotope-exchange reaction ( Vex)were estimated according to (Litwin & Wimmer, 1979)
where X = fraction of the initial [@y-'80,y-1803]GTP converted to GDP, F = fraction of positional isotope-exchange equilibrium attained in the [/3y180,~-1803]GTP pool at time t , and A, = the concentration of the initial [/3y-180,y-'*03]GTP pool. RESULTS Isotope Trapping of E-MgGTP by Rat Liver P-enolpyruvate Carboxykinase. The amount of [ c ~ - ~ ~ P ] Gtrapped TP as a function of oxaloacetate concentration in the chase was determined at each fixed concentration of [ C X - ~ ~ P I GinTthe P equilibrium mixture and analyzed as shown in Figure 1A. The upper line in Figure IA has excess enzyme ( 5 5 pM) relative
0.05
0.10
0.15
1/( [GT P*]-[G D P*]max) FIGURE
0.20
I 0.25
(1 4 .w
2: Isotope trapping from the E.M~[(u-~~P]GTP complex with
chicken liver P-enolpyruvatecarboxykinase. The experimental conditions are the same as in panels A and B in Figure 1 except that the chicken liver enzyme was present at 50 pM. to the total MgGTP concentration of 24 pM. Under these conditions, most of the MgGTP should be enzyme bound before addition of the chase solution containing oxaloacetate. This experiment therefore has an upper limit of 24 pM for GTP trapping. The values for Kb,, determined at each [CX-~~PIGTP concentration are the same within experimental error and are 40 f 10 pM. The P*,,, ([cx-~~PIGTP trapped at saturating oxaloacetate concentration) determined at each [a-32P]GTPconcentration was then analyzed in a double-reciprocal plot (Figure 1B). The dissociation constant for the E-MgGTP complex was determined to be 5 f 2 pM. The P*,,, obtained when the enzyme is saturated with Mg[a32P]GTPprior to oxaloacetate addition is equal to 35 f 3 pM, which corresponds to 0.6 f 0.1 mol of GTP trapped per enzyme subunit. As shown in Scheme I, the relative rate of M ~ [ c x - ~ ~ P ] dissociation GTP from E.M~[CY-~~P]GTP.OAA (ks) to conversion to [w3*P]GDP ( l ~ ' is~ 0.64 ~ ) f 0.05. With the kinetic constants from Table I, the rate of M ~ [ C Y - ~ ~ P ] G T P dissociation from E.Mg[a-32P]GTP ( k 2 )can be determined to be within the limits of 42 f 16 to 66 f 26 s-I. Isotope Trapping of E-MgGTP by Chicken Liver P-enolpyruvate Carboxykinase. The amount of [ C ~ - ~ ~ P ] trapped GTP with varied concentrations of oxaloacetate was determined and analyzed as shown in Figure 2A. Each line represents trapping with a fixed amount of [a-32P]GTPin the equilibrium mixture. The KbAAvalues determined at each [CX-~~PIGTP concentration are the same within the experimental errors and
Trapping and PIX with P-enolpyruvate Carboxykinases
Biochemistry, Vol. 30, No. 17, 1991 y"Phosphorus
Table I: Summary of Kinetic Constants from [c~-~~P]GTP-Trapping Experiments with P-enolpyruvate Carboxykinases rat liver chicken liver kinetic constants P*,,,(moI/mol of E) KOAA' (w M) K ' O A A(~r M ) V I C (s-I)
P-enolpyruvate carboxvkinase 0.6 i 0.1 17 f 5
40f IO
t ?
O-p-O-p3MP 0 0
0-P-0-P-GMP
Lb\
t t
0-P-0-P-GMP
L b
(s-I)
periment. %e
[y1803]GTP
1.2 f 0.2 11 f 2 16 f 5 18
18 22 13 K.ie b M ) 5 f 2 7f3 0.6 0.1 -0 k61kt14 42 & 16 to 66 f 26 26 f 9 k / (s-9 'The concentration of oxaloacetate to give half-maximum VIE,, which is determined as described in the text. bThe concentration of oxaloacetate to give half-maximum trapping of GTP. VI represents the turnover number in the direction of P-enolpyruvate formation under conditions of the experiment. V, represents the turnover number in the direction of oxaloacetate formation under conditions of the exvzd
~-"Phosphorus
? ?
P-enolpyruvate carboxvkinase
4147
is the dissociation constant for the EOMgGTP complex. Scheme I for these rate constants.
Scheme 11: Steps in the IsO-Positional Isotope Exchange for P-cnolpyruvate Carboxykinase'
I
PIX from phosphoenzyme intermediate
mtal GTP ennchmenl
90% [y.'*O,IGTP IC% [y-'8031GTp
'The upper reactions demonstrate the exchange that would occur if phosphoenzyme intermediate could be formed. The upper arm of the E represents the guanine nucleotide binding site. X is the putative enzyme nucleophile that accepts the y-phosphoryl. The lower arm of the E represents the oxaloacetate binding site. The filled oxygens represent "0.The lower reactions demonstrate the positional isotope exchange that would be expected if a series of reversible steps links with E. MgGDP.PEP.CO, complex with free MgGTP. il
are equal to 16 f 5 p M . T h e amount of [ c d 2 P ] G T P trapped a t saturating oxaloacetate concentration for each [ c d 2 P ] G T P concentration (P*max)was analyzed a s shown in Figure 2B. T h e dissociation constant for the E - M g G T P complex equals 7 f 3 p M . T h e P*,,, obtained when the enzyme is saturated with M g [ d 2 P ] G T P and oxaloacetate is 59 f 8 p M , which corresponds t o 1.2 f 0.2 mol of G T P trapped per enzyme subunit. A s shown in Scheme I, the dissociation of M g [ a 32P]GTP ( k 6 ) from E.Mg[a-32P]GTP.0AA is insignificant compared to its rate of conversion to [ c Y - ~ ~ P I G D ( lP~ ' , ~With ). the kinetic constants in Table I, the rate of dissociation of M ~ [ c x - ~ ~ P ] Gfrom T P the E . M ~ [ L Y - ~complex ~P] ( k 2 )can be estimated to be 26 f 9 s-I. Positional Isotope Exchange of [Py-i80,y-'803]GTP in the Absence of Oxaloacetate. In order to determine whether P-enolpyruvate carboxykinase is capable of forming a reversible phosphoenzyme intermediate in t h e absence of oxaloacetate, I8O positional isotope exchange was measured in the absence of the carboxylic acid. With this condition, the positional isotope exchange of [Py-'80,y-1803]GTPcan reveal any significant reversible y-phosphoryl transfer from GTP to a phosphoenzyme intermediate as shown in the upper portion of Scheme 11.
-23.6
PPM
-23.7
-23.8
PPM
FIGURE3: jlP NMR
spectra for I80-labeledGTP taken at 202.3 MHz in 40% D20at pH 9.0. The chemical shift was scaled relative to inorganic phosphate as an internal standard. The upper spectrum was determined after 90 min of the chicken liver P-enolpyruvate carboxykinase reaction in a solution containing 46 r M oxaloacetate generated from excess malate and 3.5 mM [fiy-'80,y-1803]GTPat pH 8.0 as described in the text. The lower spectrum is a control of 3.5 mM [fiy-180,y-1803]GTP from the same reaction mixture used for the upper scan, but before the reaction was initiated by the addition of enzyme. Table 11: Positional Isotope-Exchange Analysis of Chicken Liver P-enolpyruvate Carboxykinase fraction of GTP y-P of GTP [MnCI2] time enzyme' converted PO[1803]:P(rM) (min) (units) to GDPb ['8041c [OAAI (rM) 0 0
W PIX from reverse ofenryme-bound complexes
-8.20
-8.10
0 0 0 46 46 46 46 46
0 0 0 0 50 0 0 0 0 50
0 40 90 180 120 0 40 90 180 120
0 0.18 0.18 0.18 0.46 0 0.18 0.18 0.18 0.46
0 0 0 0 0 0 0.13 0.15 0.16 0.18
10:90 10:90 10:90 10:90 10:90 10:90 12:88
21:79 2575 33:69
[oxalate] (mM)
0.18 0 10:90 0 120 50 120 0.46 0 10:90 "The activity of P-enolpyruvate carboxykinase was determined in the positional isotope-exchange reaction mixture with oxaloacetate generated from malate and NAD+ by the malate dehydrogenase reaction. the absence of oxaloacetate the fraction of GTP converted into GDP was determined in 31P NMR spectra by measuring the peak corresponding to the @-phosphorusof GDP as described in the text. In the presence of oxaloacetate the fraction of GTP converted into GDP was determined by coupling to malate dehydrogenase as described in the text. 'The fraction was determined by measuring the peak area corresponding to the y-phosphorus of GTP in PO[1803]and P[180,] by using NMR at 202.3 MHz as described in the text. 0.1 0.1
T h e positional exchange of [/3y-'80,y-i803]GTP into [P180,y-1803]GTPwas determined with 31PN M R a t 202.3 M H z based on a n 180-induced 31Pchemical shift of -0.022 ppm (upfield) in the y-phosphorus and a difference of 0.012 ppm (downfield) in the P-phosphorus as shown in Figure 3. T h e experiments were carried out a t 30 OC,pH 8.0,in both the presence and absence of the V,,, activator Mn2+, a t 50 pM. As shown in Tables I1 and 111, when oxaloacetate is absent, no change in the 31Psignal of t h e y-phosphorus of G T P was observed for either chicken or r a t liver P-enolpyruvate carboxykinases. Likewise, no changes were observed in the nonbridge-'80 distribution in the @phosphorus of GTP (data not shown). T h e results indicated no positional isotope exchange of [ p y - 1 8 0 , y - 1 8 0 3 ] G T Pto [/3-180,y-i803]GTPfor
4148
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Biochemistry, Vol. 30, No. 17, 1991
Table 111: Positional Isotope-Exchange Analysis of Rat Liver P-enolpyruvate Carboxykinase with [&-180,y-1801]GTPand Excess Oxaloacetate fraction of GTP 7-P of GTP [MnCI2] time enzymea converted PO[1801]:PImin) (units) into GDPb I'8011c IuM)
[OAAI (mM) 0 0 0 0 0
0 0 0 0 50 0 0
0 40 90 180 120
0 3 6 9 20 20
0 0.1 0.1 0.1 0.12 0 0.1 0.1 0.1 0.1 0.12
0
0 0 0.07 0.14 0.20 0.34 0.60
10:90
r
Table IV: Rates of [j3y-'80,y-'*Ol]GTP Positional Isotope Exchange (PIX) Relative to [8-3H]GDP and [32P]P-enolpyruvate Exchange
[MnI (PM) 0
VpC/ Vcatb 0.040 f 0.016
VGDWTP'/
0.050
Vcat
0.01 1
VPEWTP~/V~~
0.024
0.003
0.052 f 0.004 0.033 f 0.001 0.035' a Vpixis the average value of the observed positional isotope-exchange rate, determined according to Litwin and Wimmer (1979)as described under Materials and Methods. V,, is the catalytic activity of P-enolpyruvate carboxykinase in the reaction mixture for positional isotope exchange, determined as described in the text. V G ~ P - . Gis~ Pthe initial rate of GDP to GTP exchange determined from Figure 4A. VpEp+Tp is the initial rate of P-enolpyruvate to GTP exchange determined from Figure 4B. eThe value is from a single experimental determination. 50
I
10:90 10:90 10:90 10:90
0 0 0
10:90 10:90 3 10:90 3 0 10:90 3 0 10:90 3 0 10:90 3 50 [oxalate] (mM) 0. I 0 180 0.3 0 1090 0. I 50 180 0.3 0 I090 "The activity of P-enolpyruvate carboxykinase was determined in the positional isotope-exchange reaction mixture by using the cou led pyruvate kinase/lactate dehydrogenase assay as described in the text. The fraction of GTP converted into GDP was determined in "P NMR spectra by measuring the peak corresponding to the 8-phosphorus of GDP as described in the text. CTheextent of I8O positional isotope exchange was determined by measuring the area of the peaks corresponding to the y-phosphorus of GTP in PO['801] and P['*04] by using I'P NMR at 202.3 MHz as described in the text. 3
0.5 1
positional isotope
exchange, min
0.4 1
0.3
0.2
0.1
0.0 40
50 60 70 80 positional isotope exchange, min
90
FIGURE4: Equilibrium isotopic exchange of [8-'H]GDP and ['*PIP-enolpyruvate into GTP with chicken liver P-enolpyruvate carboxykinase and oxaloacetate at 46 pM. Trace amounts of (A) [8ZH]GDP (1 pCi) or (B) [3ZP]P-enolpyruvate(0.3 pCi) were added 40 min after initiation of the positional isotope-exchange reaction. This time corresponds to the period in which I8O positional isotope exchange begins (Table 11). The concentration of [8-'H]GTP and [y-'ZP]GTP represents the exchange of labeled GDP and P-enolpyruvate into the pool of GTP, which was approximately 3.0 mM under these conditions. The reactant product concentrations were determined as described in the text.
chicken or rat liver P-enolpyruvate carboxykinases without Table V: Positional Isotope-Exchange Analysis of Chicken Liver oxaloacetate. P-enolpyruvate Carboxykinase with [/3r-'80,y-'803]GTPand Excess Positional Isotope Exchange of [ I ! ? ~ ' ~ O , ~ ' ~ Oin~the ] G T P Oxaloacetate Presence of Oxaloacetate. Positional isotope-exchange exfraction of GTP y-P of GTP periments with chicken liver P-enolpyruvate carboxykinase [OAA] [MnCI2] time enzyme" converted to PO['801]:P(mM) (rM) (min) (units) GDPb ['8041c were performed with t h e concentration of oxaloacetate 3 0 0 0 0 10:90 maintained at 46 pM by t h e equilibrium of the malate de3 0 3 0.1 0.37 10:90 hydrogenase reaction. Exchange from t h e PY-'~Obridge 3 0 6 0.1 0.41 10:90 oxygen t o t h e P-l8Oposition of GTP increased, beginning at 3 0 9.5 0.1 0.41 10:90 3 0 20 0.1 0.48 1090 40 min, a n d this was accompanied with a n increase in t h e 3 50 5 0.15 0.37 1090 amount of nonbridge l 8 0 in the @-phosphorus of GTP (Table 3 50 10 0.15 0.41 10:90 11). However, control experiments demonstrated the exchange The activity of P-enolpyruvate carboxykinase in the positional isotopeof trace amounts of [8-3H]GDP and [32P]P-enolpyruvate into exchange reaction mixture was determined with the pyruvate kinase/laceGTP coincident with positional isotope exchange. Positional tate dehydrogenase coupling enzyme assay as described in the text. "he fraction of GTP converted into GDP was determined from the "P NMR isotope exchange was detected only under conditions where spectra by measuring the peak corresponding to the 8-phosphorus of GDP both [8-3H]GDP and [32P]P-enolpyruvate exchanged into GTP as described in the text. cThe fraction was determined by measuring the (Figure 4). T h e initial rates of the product-exchange reactions area of peaks corresponding to the y-phosphorus of GTP in PO['803] and a r e within experimental error of the observed positional isotope PO[180,] by using NMR at 202.3 MHz as described in the text and Figure 3. exchange (Table 1V). Thus, the reverse reaction, after product accumulation, is responsible for the observed positional isotope no positional isotope exchange was observed even when 48% exchange. of GTP was converted t o GDP in t h e absence of Mn2+. These results were further confirmed in experiments with Similar results were obtained in the presence of 50 pM Mn2+ excess oxaloacetate in the reaction mixture. In these experiments, the oxaloacetate concentration was >20-foId above its after 41% of the GTP was converted to products. K, during the entire time course of the incubation, including Positional isotope exchange with rat liver P-enolpyruvate loss by nonenzymatic decarboxylation. As shown in Table V, carboxykinase was also determined in experiments with the
Trapping and PIX with P-enolpyruvate Carboxykinases direct addition of excess oxaloacetate. The amount of oxaloacetate added was also sufficient to maintain the concentration >20-fold above its K,,, during the time course of the reaction (Table 111). No positional isotope exchange was observed after 34% of GTP was converted to products in the absence of Mn2+ or when 60% of GTP was converted to products in the presence of 50 pM Mn2+. Positional Isotope Exchange of [&-'80,y-'803]GTP with Oxalate. Positional isotope-exchange experiments with oxalate as a nonreactive analogue of oxaloacetate were carried out with both chicken and rat liver P-enolpyruvate carboxykinases. No positional isotope exchange was observed with 1 mM oxalate for either enzyme in the presence or absence of 50 pM Mn2+ after extensive incubation (Tables I1 and 111). DISCUSSION MgCTP Trapping. Chicken and rat liver P-enolpyruvate carboxykinases trap labeled MgGTP with stoichiometries of 1.O and 0.6, respectively. These results establish that (a) bound MgGTP (or at least 60% in the case of the rat liver enzyme) is catalytically competent, (b) MgGTP can bind prior to oxaloacetate in both enzymes, since oxaloacetate is absent during the MgGTP-binding step, (c) MgGTP is released slowly from the E-MgGTP-OAA complex compared to the catalytic rate, and (d) MgGTP cannot dissociate at a kinetically significant rate from the E-MgGTP-OAA complex with chicken liver enzyme. On the basis of the complete trapping and high commitment to catalysis of MgGTP bound to the chicken enzyme, it is likely that the 60% trapping by the rat enzyme represents relative catalytic and desorption rates, rather than a mixture of 60% catalytically competent and 40% catalytically unreactive species of MgGTP. The MgGTP-trapping experiments allow determination of the partition ratio k6/k',l in Scheme I. The constant k',' includes catalysis and product release and therefore must be equal to or greater than the turnover number of 18 s-'. Since approximately 60% of the enzyme-bound MgGTP is trapped with the rat enzyme, release of MgGTP from the E. MgGTP-OAA complex (k6 in Scheme I) is in the range of 9-1 2 s-l. The limits on the rate constant for the release of MgGTP from E-MgGTP (k2in Scheme I) are 26-92 s-l, and the dissociation constant for the ESMgGTP complex, k 2 / k , , is 5 f 2 pM. From these values, the association rate of MgGTP with free enzyme ( k , in Scheme I) can be determined to be in the range of (3-20) X IO6 M-' s-l. These constants are summarized with other kinetic constants for rat liver P-enolpyruvate carboxykinase in Scheme 111. The slow release of MgGTP from the ternary complex and the resultant partial trapping of the nucleotide are consistent with the steady-state random kinetic mechanism proposed previously (Jomain-Baum & Schramm, 1978). The rate constant of (3-20) X lo6 M-I s-I for EaMgGTP formation is somewhat smaller than the diffusion limit of -IO* M-' s-' for a protein and substrate the sizes of P-enolpyruvate carboxykinase and MgGTP. Thus, rapid structural changes are likely to occur in concert with MgGTP binding. Limits on the release rate of MgGTP from the E-MgGTP complex indicate that release is slow, accounting for the relatively tight dissociation constant of 5 pM. The ability to trap 60% of the enzyme-bound MgGTP from the E-MgGTPSOAA complex in the presence of a saturating concentration of oxaloacetate and the slow release of MgGTP from this complex establish that the presence of oxaloacetate hinders MgGTP release. Trapping of MgGTP with the enzyme from chicken liver is consistent with an ordered mechanism with MgGTP binding first. The nucleotide is unable to dissociate from the enzyme
Biochemistry, Vol. 30, No. 17, 1991
4149
in the presence of bound oxaloacetate. Thus, all bound MgGTP is converted to product when excess oxaloacetate is present. MgGTP binds free enzyme with a rate constant lower than the diffusion limit, and dissociation is slow, resulting in a dissociation constant of 7 pM. Although a very slow rate of MgGTP release from the E-MgGTPnOAA complex would not have been detected in these experiments, a rate of 1 s-I would have been observed. Thus, at saturating oxaloacetate, enzyme-bound MgGTP is completely committed to catalysis. These results are consistent with the steady-state kinetic proposal of Felicioli et al. (1 970) for a steady-state ordered mechanism for the chicken enzyme with ITP being added prior to oxaloacetate. Product release was proposed to occur in the order C 0 2 ,P-enolpyruvate, and IDP. Apart from the extent of catalytic commitment of MgGTP, the kinetic and rate constants of the chicken and rat enzymes are similar (Scheme 111). Thus, release of MgGTP from the EmMgGTP complex for the chicken enzyme is also slow at 26 s-l. NMR studies with the chicken enzyme and MgGTP in the presence of enzyme-bound Mn2+ have provided values of greater than 2.1 X 1O1O M-' s-' for association of the enzyme-Mn2+ complex with MgGTP and 4 X lo4 s-, for dissociation of MgGTP from the complex (Lee & Nowak, 1984). These values differ substantially from the results reported here and the normal assumptions for diffusion-controlled association rates of 108-109 M-' s-l (Eigen & Hammes, 1963). The distances between the enzyme-bound Mn2+and the a-,0-, and y-phosphates of MgGTP were reported to be 5-6 A on the basis of the rapid-exchange assumption for MgGTP. These values may need to be reevaluated on the basis of the relatively slow release of MgGTP from the enzyme-Mn2+ complex shown in Scheme 111. [p,7-'80] GTP Positional Isotope Exchange in the Absence of Oxaloacetate. In the absence of oxaloacetate, neither the chicken nor the rat liver P-enolpyruvate carboxykinases were capable of causing exchange from the ~ , Y - ' ~ bridge O oxygen to the & l 8 0 position. This exchange should occur if the enzyme catalyzes a GTP + GDP exchange as previously reported for the rat liver enzyme with Mn2+as the sole divalent cation (Jomain-Baum & Schramm, 1978; see Scheme 11). Attempts to repeat the GTP + GDP exchange with the highly purified P-enolpyruvate carboxykinases used in this study gave no exchange with MgGTP as substrate. Thus, the previous result is likely due to an enzymatic impurity that catalyzes GTP + GDP exchange or is catalyzed by P-enolpyruvate carboxykinase when both required divalent metals are Mn2+. The results of the positional isotope-exchange experiments reported here establish that any such enzymatic phosphorylation is not part of the required catalytic cycle for P-enolpyruvate carboxykinase when MgGTP is used as a substrate with Mn2+ as activator. The transfer of the y-phosphoryl group of nucleotide triphosphates occurs with stereochemical inversion for both the guinea pig mitochondrial and the rat cytosolic P-enolpyruvate carboxykinases (Sheu et al., 1984; Konopka et al., 1986). These reports are consistent with the lack of positional isotope exchange reported here in the absence of oxaloacetate. A requirement for phosphoryl transfer could be occupation of the binding site for the carboxylic acid. Addition of oxalate, a competitive inhibitor with respect to oxaloacetate with a Ki value of
